1. Field of the Invention
The present invention relates to isolated proteases of the RP-II type and variants of RP-II proteases exhibiting improved properties in comparison to the parent RP-II protease, DNA constructs and vectors coding for the expression of said proteases and variants, host cells capable of expressing the proteases and variants from the DNA constructs, as well as a method of producing them by cultivating said host cells. The proteases may advantageously be used as constituents in detergent compositions and additives.
2. Description of Related Art
Proteases of the subtilisin family (Siezen et al., 1997, Protein Science, 6: 501–523) have been used in the detergent industry for many years due to their superiority over other protease types.
A large number of subtilisins and the related subtilases are known.
Protease variants have been produced in a number of subtilisin proteases in order to provide changes in various properties, such as thermo stability, specific activity, pH-dependency, isoelectric point, wash performance, oxidation stability, autoproteolysis, etc.
Such variants are disclosed in various patent publications, such as EP 130 756, EP 251 446, and EP 824 585.
The fact that detergents constantly are being developed to satisfy various user demands provides an incentive to continuously develop proteases capable of providing excellent performance in detergents.
Bacillus proteases of the RP-II type are another type of serine proteases that in primary structure are similar to chymotrypsinogen.
The first description of a protease of the RP-II family of Bacillus proteases was in U.S. Pat. No. 4,266,031 (Tang et al., Novo Industri A/S), where it was designated Component C and tentatively characterized as not being a serine protease or metalloprotease. Component C was considered a contaminant in the production of the Bacillus licheniformis alkaline protease, subtilisin Carlsberg.
EP 369 817 (Omnigene Bioproducts, Inc.) identifies the B. subtilis member of the RP-II family by its amino acid and DNA sequences. The enzyme was stated not to be a serine protease, and the family name RP-II designated (Residual Protease II). The enzyme was characterized further as a metalloprotease by the inventors of EP 369 817 (Rufo et al., 1990, J. Bacteriol., 2: 1019–1023, and Sloma et al., 1990, J. Bacteriol., 2: 1024–1029), designating the enzyme as mpr.
WO 91/13553 (Novo Nordisk A/S) discloses the amino acid sequence of the C component and states that it is a serine protease specific for glutamic and aspartic acid, while EP 482 879 (Shionogi & Co. Ltd.) discloses the enzyme and a DNA sequence encoding the C component from B. licheniformis ATCC No. 14580, naming the enzyme BLase. EP 482 879 describes the protease as being specific for glutamic acid.
Okamoto et al. (Appl. Microbiol. Biotechnol., 1997, 48: 27–33) disclose that the B. subtilis homologue of BLase, named BSase was identical to the above-mentioned enzyme, mpr/RP-II.